In Vivo Low-Temperature Plasma Ionization Mass Spectrometry (LTP-MS) Reveals Regulation of 6-Pentyl-2H-Pyran-2-One (6-PP) as a Physiological Variable during Plant-Fungal Interaction

Volatile organic compounds (VOCs) comprises a broad class of small molecules (up to ~300 g/mol) produced by biological and non-biological sources. VOCs play a vital role in an organism's metabolism during its growth, defense, and reproduction. The well-known 6-pentyl-α-pyrone (6-PP) molecule is an example of a major volatile biosynthesized by Trichoderma atroviride that modulates the expression of PIN auxin-transport proteins in primary roots of Arabidopsis thaliana during their relationship. Their beneficial relation includes lateral root formation, defense induction, and increased plant biomass production. The role of 6-PP has been widely studied due to its relevance in this cross-kingdom relationship. Conventional VOCs measurements are often destructive; samples require further preparation, and the time resolution is low (around hours). Some techniques enable at-line or real-time analyses but are highly selective to defined compounds. Due to these technical constraints, it is difficult to acquire relevant information about the dynamics of VOCs in biological systems. Low-temperature plasma (LTP) ionization allows the analysis of a wide range of VOCs by mass spectrometry (MS). In addition, LTP-MS requires no sample preparation, is solvent-free, and enables the detection of 6-PP faster than conventional analytical methods. Applying static statistical methods such as Principal Component Analysis (PCA) and Discriminant Factorial Analysis (DFA) leads to a loss of information since the biological systems are dynamic. Thus, we applied a time series analysis to find patterns in the signal changes. Our results indicate that the 6-PP signal is constitutively emitted by T. atroviride only; the signal shows high skewness and kurtosis. In A. thaliana grown alone, no signal corresponding to 6-PP is detected above the white noise level. However, during T. atroviride-A. thaliana interaction, the signal performance showed reduced skewness and kurtosis with high autocorrelation. These results suggest that 6-PP is a physiological variable that promotes homeostasis during the plant-fungal relationship. Although the molecular mechanism of this cross-kingdom control is still unknown, our study indicates that 6-PP has to be regulated by A. thaliana during their interaction.